RESUMEN
The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.
RESUMEN
Oncomodulin (Ocm) is a myeloid cell-derived growth factor that enables axon regeneration in mice and rats after optic nerve injury or peripheral nerve injury, yet the mechanisms underlying its activity are unknown. Using proximity biotinylation, coimmunoprecipitation, surface plasmon resonance, and ectopic expression, we have identified armadillo-repeat protein C10 (ArmC10) as a high-affinity receptor for Ocm. ArmC10 deletion suppressed inflammation-induced axon regeneration in the injured optic nerves of mice. ArmC10 deletion also suppressed the ability of lesioned sensory neurons to regenerate peripheral axons rapidly after a second injury and to regenerate their central axons after spinal cord injury in mice (the conditioning lesion effect). Conversely, Ocm acted through ArmC10 to accelerate optic nerve and peripheral nerve regeneration and to enable spinal cord axon regeneration in these mouse nerve injury models. We showed that ArmC10 is highly expressed in human-induced pluripotent stem cell-derived sensory neurons and that exposure to Ocm altered gene expression and enhanced neurite outgrowth. ArmC10 was also expressed in human monocytes, and Ocm increased the expression of immune modulatory genes in these cells. These findings suggest that Ocm acting through its receptor ArmC10 may be a useful therapeutic target for nerve repair and immune modulation.
Asunto(s)
Axones , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Ganglios Espinales/metabolismo , Regeneración Nerviosa , Proyección Neuronal , Células Receptoras SensorialesRESUMEN
Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+ microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENT Despite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.
Asunto(s)
Axones/metabolismo , Complemento C1q/metabolismo , Complemento C3/metabolismo , Células Mieloides/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Axones/inmunología , Complemento C1q/inmunología , Complemento C3/inmunología , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/inmunología , Regeneración Nerviosa/fisiología , Traumatismos del Nervio Óptico/inmunología , Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/inmunologíaRESUMEN
The optic nerve conveys information about the outside world from the retina to multiple subcortical relay centers. Until recently, the optic nerve was widely believed to be incapable of re-growing if injured, with dire consequences for victims of traumatic, ischemic, or neurodegenerative diseases of this pathway. Over the past 10-20 years, research from our lab and others has made considerable progress in defining factors that normally suppress axon regeneration and the ability of retinal ganglion cells, the projection neurons of the retina, to survive after nerve injury. Here we describe research from our lab on the role of inflammation-derived growth factors, suppression of inter-cellular signals among diverse retinal cell types, and combinatorial therapies, along with related studies from other labs, that enable animals with optic nerve injury to regenerate damaged retinal axons back to the brain. These studies raise the possibility that vision might one day be restored to people with optic nerve damage.
Asunto(s)
Regeneración Nerviosa/fisiología , Traumatismos del Nervio Óptico/metabolismo , Nervio Óptico/fisiología , Células Ganglionares de la Retina/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Humanos , Mediadores de Inflamación/metabolismo , Nervio Óptico/ultraestructura , Traumatismos del Nervio Óptico/patología , Células Ganglionares de la Retina/ultraestructuraRESUMEN
The inflammatory response to spinal cord injury (SCI) involves localization and activation of innate and adaptive immune cells and proteins, including the complement cascade. Complement C3 is important for the classical, alternative, and lectin pathways of complement activation, and its cleavage products C3a and C3b mediate several functions in the context of inflammation, but little is known about the potential functions of C3 on regeneration and survival of injured neurons after SCI. We report that 6 weeks after dorsal hemisection with peripheral conditioning lesion, C3-/- mice demonstrated a 2-fold increase in sensory axon regeneration in the spinal cord in comparison to wildtype C3+/+ mice. In vitro, addition of C3 tripled both myelin-mediated neurite outgrowth inhibition and neuron loss versus myelin alone, and ELISA experiments revealed that myelin serine proteases cleave C3 to generate active fragments. Addition of purified C3 cleavage products to cultured neurons suggested that C3b is responsible for the growth inhibitory and neurotoxic or anti-adhesion activities of C3. These data indicate that C3 reduces neurite outgrowth and neuronal viability in vitro and restricts axon regeneration in vivo, and demonstrate a novel, non-traditional role for this inflammatory protein in the central nervous system.
Asunto(s)
Axones/metabolismo , Complemento C3/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Complemento C3/deficiencia , Ganglios Espinales/patología , Ratones Noqueados , Vaina de Mielina/metabolismo , Regeneración Nerviosa , Neuritas/metabolismo , Nervio Ciático/lesiones , Nervio Ciático/patología , Serina Proteasas/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/cirugíaRESUMEN
Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI.
Asunto(s)
Axones/efectos de los fármacos , Complemento C1q/farmacología , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Neuritas/fisiología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Arginasa/genética , Arginasa/metabolismo , Células Cultivadas , Complemento C1q/genética , Complemento C1q/metabolismo , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteína GAP-43/metabolismo , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/fisiología , Glicoproteína Asociada a Mielina/metabolismo , Neuritas/efectos de los fármacos , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/patología , Traumatismos de la Médula Espinal/patologíaRESUMEN
The pathology associated with spinal cord injury (SCI) is caused not only by primary mechanical trauma, but also by secondary responses of the injured CNS. The inflammatory response to SCI is robust and plays an important but complex role in the progression of many secondary injury-associated pathways. Although recent studies have begun to dissect the beneficial and detrimental roles for inflammatory cells and proteins after SCI, many of these neuroimmune interactions are debated, not well understood, or completely unexplored. In this regard, the complement cascade is a key component of the inflammatory response to SCI, but is largely underappreciated, and our understanding of its diverse interactions and effects in this pathological environment is limited. In this review, we discuss complement in the context of SCI, first in relation to traditional functions for complement cascade activation, and then in relation to novel roles for complement proteins in a variety of models.